High cell density-mediated pericellular hypoxia is a crucial factor inducing expression of the intrinsic hypoxia marker CA IX in vitro in HeLa cells

Oxygen plays a central role in respiration of the cells and thus in generation of energy by aerobic metabolism. The cells precisely detect oxygen level and changes in oxygen perfusion leads to induction of various responses enabling to adapt to unfavorable conditions. CA IX carbonic anhydrase is a hypoxia-inducible tumor-associated antigen which is overexpressed in dense HeLa cells. Presented study investigates the effects of oxygen tension on CA IX expression in HeLa cell culture. Using of an immunoradiometric assay to quantify CA IX protein, it was revealed that expression of CA IX correlates with increasing cell density, lactate production and medium acidification under normoxic conditions. These observations and hypoxia-inducibility of CA IX suggested a possible role of pericellular hypoxia in density-induced CA IX expression. To test this hypothesis, HeLa cells were incubated in normobaric hyperoxia (50% O2) or cell culture medium was convected to disturb oxygen deprivation. Both approaches completely abrogated CA IX expression in dense HeLa cell cultures and therefore confirmed the importance of decreased oxygen tension in high cell density-induced CA IX expression. In addition, HeLa cells exposed to hyperoxia retained inducibility of CAIX expression by transition metals and iron chelators, suggesting that they act independently of cell density mediated-pO2-gradient or at a downstream site from oxygen sensor. Observed data indicate that high cell density-lowered pericellular pO2 is a crucial factor inducing CA IX expression and influencing composition of metabolic micromilieu surrounding the dense HeLa cells.